Characterization of progressive metaplasia in the gastric corpus mucosa of Mongolian gerbils infected with Helicobacter pylori

Spasmolytic polypeptide‐expressing metaplasia (SPEM) and intestinal metaplasia are considered neoplastic precursors of gastric adenocarcinoma in humans. Loss of parietal cells causes the development of SPEM in the gastric corpus and then chronic inflammation drives SPEM toward a more proliferative lineage. Mongolian gerbils infected with Helicobacter pylori develop chronic gastritis and metaplasia, mimicking aspects of human gastritis with H. pylori infection. We therefore examined metaplastic lineages in the gastric corpus mucosa of gerbils infected by H. pylori strain 7.13, which produces rapid onset of severe inflammation. Six weeks following H. pylori infection, Griffonia simplicifolia lectin II (GSII)‐positive SPEM developed in the base of oxyntic glands in association with parietal cell loss and inflammation. In association with severe inflammation, SPEM glands evolved into aberrant phenotypes, including branched lesions, dilated lesions, and penetrating invasive glands. Mucin 4 (MUC4) was up‐regulated in SPEM and progressive SPEM. Clusterin was expressed in the tips of branched and dilated lesions and throughout regions of invasive glands. Intriguingly, clusterin‐positive regions in these lesions expressed Ki67 and matrix metalloproteinase 7 (MMP‐7). These same regions were also positive for expression of phospho‐IkBα, suggestive of activated NFkB signalling. These findings suggest that clusterin‐positive regions in progressive phenotypes of SPEM have invasive characteristics. Thus, H. pylori infection in gerbils induces SPEM, which then can progress to further aberrant and invasive metaplastic phenotypes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Colonization and inflammation deficiencies in Mongolian gerbils infected by Helicobacter pylori chemotaxis mutants

Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in the tlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved in H. pylori pathogenesis.     

Association of spasmolytic polypeptide-expressing metaplasia with carcinogen administration and oxyntic atrophy in rats

Spasmolytic polypeptide (TFF2)-expressing metaplasia (SPEM) is a gastric metaplastic lineage associated with the development of intestinal-type gastric adenocarcinoma. To study the etiology of this potential neoplastic precursor metaplasia, we used surgical rat models of remnant gastric adenocarcinoma studied with and without exposure to nitroso carcinogen. Animals with truncal vagotomy without duodenogastric reflux procedures demonstrated normal mucous neck cell spasmolytic polypeptide (SP) immunostaining. In these animals, anti-proliferating cell nuclear antigen (PCNA)-labeled nuclei were found in the normal midgland progenitor zone. Rats that received anatomic alterations that augmented the degree of duodenogastric reflux, however, revealed expansion of basally placed SP immunoreactive cells with early phenotypic changes of SPEM. Seventy percent of animals with antrectomy and carcinogen (with or without vagotomy) developed SPEM at the base of the gastric mucosa. In association with the appearance of this metaplastic lineage, a distinct second zone of PCNA-labeled nuclei developed in the deepest portion of the mucosa. Of interest, three animals demonstrating these changes developed intestinal-type gastric adenocarcinoma. Finally, we studied the immunostaining pattern of intrinsic factor, normally a chief cell marker in rat fundic mucosa. In animals with SPEM, we observed coexpression of SP and intrinsic factor in SPEM cells at the base of the mucosa. These findings support our hypothesis that SPEM develops from a second progenitor cell population, reflecting either the unmasking of a cryptic zone or transdifferentiation of chief cells.     

Serum immunoglobulin G immune response to Helicobacter pylori antigens in Mongolian gerbils

The Mongolian gerbil model for Helicobacter pylori infection is an animal model that mimics human disease. We examined the serum immune response to H. pylori infection in gerbils by enzyme-linked immunosorbent assay (ELISA) and Western blotting, both with whole-cell (H. pylori) extracts. A total of 66 7-week-old specific-pathogen-free male gerbils were inoculated orogastrically with H. pylori strain ATCC 43504. Sera were collected 1, 2, 4, 8, 12, 26, 38, and 52 weeks after H. pylori inoculation. Sixty-nine noninfected gerbils and their sera were used as controls. The specificity of the ELISA was 95.7%. The frequency of seropositivity increased over time: 2 of 10 (20%), 7 of 10 (70%), and 7 of 7 (100%) samples of sera from inoculated gerbils were positive for H. pylori at 2, 4, and 8 weeks postinoculation, respectively. Western blot assays showed that the primary immunoglobulin G (IgG) response against low-molecular-mass (25-, 30-, and 20-kDa) proteins appeared after a lag period of 2 to 8 weeks after inoculation. Antibodies against 160-, 150-, 110-, 120-, 80-, 66-, and 63-kDa proteins were observed 12 weeks after inoculation. The early reactive 30-kDa protein was identified as a urease alpha subunit by N-terminal amino acid sequencing. After 26 weeks, two groups of animals could be distinguished: one group developed ulcers (n = 5), and the other developed hyperplastic polyps without ulcers (n = 19). Gerbils in the gastric ulcer group showed significantly higher serum anti-H. pylori IgG levels than did gerbils in the hyperplastic group (P = 0.001) as measured by ELISA. Furthermore, a higher proportion of animals developed antibodies to H. pylori proteins of 26, 25, and 20 kDa in the ulcer group than those animals with hyperplastic polyps (75 to 100% versus 17 to 50%) in Western blot assays. These results highlight the importance of the immune response of the host in the development of H. pylori-related gastric lesions.     

Comparative analysis of gastric bacterial microbiota in Mongolian gerbils after long-term infection with Helicobacter pylori

Quantitative (qt) real time PCR using 16SrDNA primers is useful for determination of the bacterial composition of the gastric microbiota in Mongolian gerbils. The aim of this study was to determine the change in the gastric microbiota after long-term infection with Helicobacter pylori. One year after inoculation with H. pylori, five gerbils were determined as H. pylori-positive and 6 gerbils H. pylori-negative by culture and real time qt PCR methods. The gastric microbiota of each group of gerbils was also compared with that of 6 gerbils uninfected with H. pylori. DNA from the Atopobium cluster, Bifidobacterium spp., Clostridium coccoides group, Clostridium leptum subgroup, Enterococcus spp. and Lactobacillus spp. were detected in the gastric mucus of both infected and uninfected gerbils. In contrast, Eubacterium cylindroides group and Prevotella spp. were detected only in H. pylori-negative gerbils. The numbers of C. leptum subgroup, C. coccoides group and Bifidobacterium spp. in gastric mucus of H. pylori-negative Mongolian gerbils were significantly lower than those in non-infected gerbils. The results obtained suggest that the composition of gastric indigenous microbiota in Mongolian gerbils may be disturbed by long-term infection with H. pylori, and that these changes may in fact inhibit H. pylori infection.     

Helicobacter pylori-induced chronic active gastritis, intestinal metaplasia, and gastric ulcer in Mongolian gerbils

The establishment of persisting Helicobacter pylori infection in laboratory animals has been difficult, but in 1996 Hirayama reported the development of a successful Mongolian gerbil model. The present study was undertaken with two aims: to better characterize the normal histological structure and histochemical properties of the gastric mucosa of the Mongolian gerbil; and to evaluate the progression of the histopathological features of H. pylori-induced gastritis in this animal model for one year after the experimental infection. Seventy-five Mongolian gerbils were used. Mongolian gerbils were sacrificed at 2, 4, 8, 12, 26, 38, and 52 weeks after H. pylori inoculation. Sections prepared from stomachs immediately fixed in Carnoy's solution were stained with hematoxylin and eosin and Alcian blue at pH 2.5/periodic acid-Schiff, a dual staining consisting of the galactose oxidase-cold thionin Schiff reaction and paradoxical Concanavalin A staining, and with immunostaining for H. pylori and BrdU. H. pylori infection induced in the Mongolian gerbil a chronic active gastritis, in which a marked mucosal infiltration of neutrophils on a background of chronic inflammation became detectable 4 weeks after inoculation and continued up to 52 weeks. Intestinal metaplasia and gastric ulcers appeared after 26 weeks in some of the animals, whereas others developed multiple hyperplastic polyps. The Mongolian gerbil represents a novel and useful model for the study of H. pylori-induced chronic active gastritis and may lend itself to the investigation of the epithelial alterations that lead to intestinal metaplasia and gastric neoplasia.     

幽门螺杆菌诱导蒙古沙鼠胃类癌发生的实验研究

目的 探讨幽门螺杆菌(Hp)感染在蒙古沙鼠胃类癌发生中的作用及Hp去除后对胃类癌发生的影响.方法 100只蒙古沙鼠分成7组,A、B为空白对照组,C、D、E、F、G为Hp感染组,其中F、G组Hp感染后再去除.结果 空白对照组(A、B组)未见ECL细胞增生/异型增生及类癌形成,Hp感染后(C、D、E组),血清抗Hp IgG抗体、胃泌素水平均明显增高(P<0.01),ECL细胞增生/异型增生及类癌发生率分别为27.8%(5/18)、31.2%(5/16)、58.3%(14/24)和16.7%(3/18)、31.2%(5/16)、62.5%(15/24),均明显高于对照组(P<0.01),随着Hp感染时间的延长,ECL病变面积明显增加(P<0.01);Hp去除后,血清抗Hp ISG抗体、胃泌素水平明显降低,ECL细胞增生/异型增生及类癌的发生率降低,分别为25.0%(4/16)、15.4%(2/13)和37.5%(6/16)、23.1%(3/13).早期阶段去除Hp(G组),ECL病变发生率及类癌的面积明显低于非去除组(E组)(P<0.001).血清抗Hp ISG抗体与胃泌素水平及胃泌素水平与胃类癌的发生呈正相关(P<0.001).结论 Hp感染在蒙古沙鼠胃类癌发生中起重要作用,去除Hp可有效预防蒙古沙鼠胃类癌的发生.     

Effects of Helicobacter pylori infection on the link between regenerating gene expression and serum gastrin levels in Mongolian gerbils

Although regenerating gene (Reg) protein is reported to have a trophic effect on gastric epithelial cells, its involvement in human gastric diseases is not clear. We have recently shown that both gastrin and gastric mucosal inflammation enhance Reg gene expression in the fundic mucosa in rats. This study was designed to clarify whether Reg protein is involved in Helicobacter pylori-induced gastritis and whether Reg gene expression is linked to serum gastrin levels in this condition. Mongolian gerbils were inoculated with an H. pylori strain isolated from a gastric cancer patient. Four weeks later, some of the gerbils with H. pylori infection were eradicated by lansoprazole, amoxicillin, and clarithromycin. The time courses of changes in Reg gene expression, serum gastrin levels, gastric acidity, and histopathologic factors were examined. Four weeks after H. pylori infection, gastritis started spreading to the fundic mucosa, and gastric acidity started reducing. Serum gastrin levels and Reg mRNA expression in the fundus were significantly increased 6 weeks after infection. Reg mRNA expression in the fundus correlated significantly with both serum gastrin levels and the severity of fundic mucosal inflammation. After H. pylori eradication, serum gastrin levels and fundic mucosal inflammation were normalized, and the increase in Reg mRNA expression was abolished. The Reg gene is associated with hypergastrinemia and fundic mucosal inflammation and may be involved in H. pylori-induced gastritis.     

Structure of lamina propria lymphoid follicles and associated epithelium in the gastric mucosa during Helicobacter pylori infection in ulcer-bearing Mongolian gerbils

To develop a gerbil model of Helicobacter pylori-induced chronic active gastritis comparable in severity to human lesions, we made acetic acid-induced ulcer in the anterior antral wall and concurrently challenged 1 x 10(8) colony-forming units bacteria per os. At 30 and 60 days after inoculation, the number of viable bacteria colonizing on the surface epithelium of the gastric mucosa was larger in ulcer-bearing animals compared to non-bearing ones. Furthermore, in the former animals, neutrophil and mononuclear cell infiltration as well as lymphoid follicle formation in the lamina propria was more prominent. Electron microscopically, lymphoid follicle-associated epithelium displayed specialized structures. Namely, brush cells interposed between mucous epithelial cells and characterized by prominent microfilament bundles and many apical vesicles or caveola specifically embraced the cluster of intraepithelially invading lymphocytes and macrophage-like cells by the attenuated cytoplasm in an analogous manner to M cells in Peyer's patches. The present study has demonstrated that ulcer formation enhances both H. pylori colonization and lamina propria lymphoid follicle formation and suggested that follicle-associated epithelium might play roles in the delivery of intraluminal antigen.     

Experimental Infection of Mongolian Gerbils with Wild-Type and Mutant Helicobacter pylori Strains

Experimental Helicobacter pylori infection was studied in Mongolian gerbils with fresh human isolates that carry or do not carry cagA (cagA-positive or cagA-negative, respectively), multiply passaged laboratory strains, wild-type strain G1.1, or isogenic ureA, cagA, or vacA mutants of G1.1. Animals were sacrificed 1 to 32 weeks after challenge, the stomach was removed from each animal for quantitative culture, urease test, and histologic testing, and blood was collected for antibody determinations. No colonization occurred after ≥20 in vitro passages of wild-type strain G1.1 or with the ureA mutant of G1.1. In contrast, infection occurred in animals challenged with wild-type G1.1 (99 of 101 animals) or the cagA (25 of 25) or vacA (25 of 29) mutant of G1.1. Infection with G1.1 persisted for at least 8 months. All 15 animals challenged with any of three fresh human cagA-positive isolates became infected, in contrast to only 6 (23%) of 26 animals challenged with one of four fresh human cagA-negative isolates (P < 0.001). Similar to infection in humans, H. pylori colonization of gerbils induced gastric inflammation and a systemic antibody response to H. pylori antigens. These data confirm the utility of gerbils as an animal model of H. pylori infection and indicate the importance of bacterial strain characteristics for successful infection.     

Assay of urease-inhibiting activity in serum from children infected with Helicobacter pylori.

In order to provide a basis for obtaining further information concerning the host response to Helicobacter pylori urease, four assay methods for detecting urease-inhibiting activity in serum were examined. A quantitative assay, established in a COBAS BIO centrifugal fast analyzer and based on detection of the consumption of NADH by glutamate dehydrogenase stimulated by ammonia production, was considered most suitable for large-scale serological work. Serum samples from 63 children (aged 5 to 16 years), 28 of whom had seropositive H. pylori gastritis, were assayed. One of the serum samples in this latter group showed significant inhibitory activity. This serum sample was one of 13 in the seropositive group known to bind to urease antigen. It showed no inhibitory activity against Bacillus pasteurii or jack bean urease. Protein A binding and heat treatment indicated that the inhibitory activity was immunoglobulin G mediated. The patient from whom this sample was collected showed no distinctive features in his illness. The COBAS BIO analyzer-based urease inhibition assay provides a new tool for studying one aspect of the host response to H. pylori infection.     

The effect of Helicobacter felis and Helicobacter bizzozeronii on the gastric mucosa in Mongolian gerbils: a sequential pathological study

In contrast to Helicobacter(H.) pylori, little is known about the pathogenic mechanisms of gastric non-H. pylori Helicobacter species. Mongolian gerbils were inoculated intragastrically with H. felis or H. bizzozeronii and killed at different timepoints post-inoculation (p.i.), stomach tissue being taken for light and transmission electron microscopy (TEM) and polymerase chain reaction (PCR) analysis. Parietal cells (PCs), apoptosis, cell proliferation and nuclear factor-kappaB (NF-kappaB) activation were "visualized" immunohistochemically. Inflammation consisted of neutrophilic granulocytes, mainly in the antrum, and lymphocytic infiltrates around the limiting ridge and throughout the stomach mucosa and submucosa. From day 11 p.i. onwards, H. felis-inoculated animals showed moderate to severe loss of PCs extending from the limiting ridge into the fundus. Apoptotic cells, spiral bacteria, cell proliferation, and NF-kappaB activation were detected at the transition zone between affected and normal PCs. TEM revealed interaction of H. felis flagella with PCs and chief cells. Moreover, H. felis was seen in proximity to, and inside, necrotic cells. At 10 weeks p.i., some H. felis-infected gerbils showed complete loss of fundic glands, and mucous metaplasia of the epithelium. H. bizzozeronii, which made no flagellar contact with epithelial cells, was associated with only mild PC loss. The mechanism by which H. felis induces PC necrosis and apoptosis remains unclear. The observed flagellar contact and NF-kappaB activation may play an important role in H. felis-associated inflammation.